Hydroxyindoles, their use as inhibitors of phosphodiesterase 4 and processes for their preparation

ABSTRACT

The invention relates to new hydroxyindoles of the Formula,their use as inhibitors of phosphodiesterase 4 and processes for their preparation.

This application is a continuation of Ser. No. 09/653,685 filed Sep. 1,2000 abandoned which is a division of Ser. No. 09/300,973 filed Apr. 28,1999 now U.S. Pat. No. 6,251,923.

FIELD OF THE INVENTION

The present invention relates to novel, substituted hydroxyindolesprocesses for their preparation, pharmaceutical preparations containingthese compounds, and a method for the use of these compounds which arephosphodiesterase 4 inhibitors, as active compounds for the treatment ofdisorders which can be affected by inhibition of phosphodiesterase 4activity in immunocompetent cells (e.g. macrophages and lymphocytes).

BACKGROUND

The activation of cell membrane receptors by transmitters leads to theactivation of the second messenger system. Adenylate cyclase synthesizesactive cyclic AMP (cAMP) or cyclic GMP (cGMP) from AMP and GMP. Theselead, for example, to relaxation in smooth muscle cells or to inhibitionof mediator release or synthesis in inflammatory cells. The breakdown ofthe second messenger cAMP and cGMP is carried out by thephosphodiesterases (PDE). To date, 7 families of PDE enzymes (PDE1-7)are known, which differ by their substrate specificity (cAMP, cGMP orboth) and the dependence on other substrates (e.g. calmodulin). Theseisoenzymes have different functions in the body and are prominent todifferent extents in the individual cell types (Beave J A, Conti M andHeaslip R J, Multiple cyclic nucleotide phosphodiesterases, Mol.Pharmacol. 1994, 46: 399-405; Hall IP, Isoenzyme selectivephosphodiesterase inhibitors; potential clinical uses, Br. J. clin.Pharmacol. 1993, 35: 1-7). As a result of inhibition of the various PDEisoenzyme types, there is an accumulation of cAMP or cGMP in the cells,which can be therapeutically utilized (Torphy T J, Livi G P, ChristensenS B, Novel Phosphodiesterase Inhibitors for the Therapy of Asthma, DrugNews and Perspectives 1993, 6: 203-214).

In the cells important for allergic inflammation (lymphocytes, mastcells, eosinophilic granulocytes, macrophages), the prevailing PDEisoenzyme is of type 4 (Torphy, J T. and Undem, B. J., Phosphodiesteraseinhibitors: new opportunities for the treatment of asthma, Thorax 1991,46: 512-523). The inhibition of PDE 4 by suitable inhibitors istherefore considered as an important starting point for the therapy of alarge number of allergically induced disorders (Schudt Ch, Dent G, RabeK, Phosphodiesterase Inhibitors, Academic Press London 1996).

An important property of phosphodiesterase 4 inhibitors is theinhibition of the release of tumour necrosis factor α (TNFα) frominflammatory cells. TNFα is an important pro-inflammatory cytokine,which affects a large number of biological processes. TNFα is released,for example, from activated macrophages, activated T lymphocytes, mastcells, basophils, fibroblasts, endothelial cells and astrocytes in thebrain. It has a self-activating effect on neutrophils, eosinophils,fibroblasts and endothelial cells, as a result of which varioustissue-destroying mediators are released. In monocytes, macrophages andT lymphocytes, TNFα brings about the increased production of furtherpro-inflammatory cytokines such as GM-CSF (granulocyte-macrophagecolony-stimulating factor) or interleukin-8. TNFα plays a central partdue to its inflammation-promoting and catabolic action in a large numberof disorders, such as inflammation of the airways, inflammation of thejoints, endotoxic shock, tissue rejection, AIDS and numerous otherimmunological disorders. Inhibitors of phosphodiesterase 4 are thus alsosuitable for the therapy of disorders of this type which are associatedwith TNFα.

Chronic obstructive pulmonary diseases (COPD) are widespread in thepopulation and also have great economic importance. Thus COPD diseasescause about 10-15% of all illness costs in the developed countries andabout 25% of all cases of death in the USA are to be attributed to thiscause (Norman P.: COPD: New developments and therapeutic opportunities,Drug News Perspect. 11 (7), 431-437, 1998), however the patients at thetime of death are usually over 55 years old (Nolte D.: ChronischeBronchitis—eine Volkskrankheit multifaktorieller Genese.Atemw.-Lungenkrkh. [Chronic bronchitis—a widespread disease ofmultifactorial origin]. 20 (5), 260-267, 1994). The WHO estimates thatCOPD will be the third most frequent cause of death within the next 20years.

The syndrome of chronic obstructive lung diseases (COPD) summarizesvarious syndromes of chronic bronchitis with the symptoms coughing andexpectoration and progressive and irreversible impairment of lungfunction (exhalation is particularly affected). The course of thedisease is episodic and often complicated by bacterial infections(Rennard S. I.: COPD: Overview of definitions, Epidemiology, and factorsinfluencing its development. Chest, 113 (4) Suppl., 235S-241S, 1998). Inthe course of the disease, the lung function continuously decreases, thelungs become increasingly emphysematous and the respiratory distress ofthe patients is obvious. This disease clearly adversely affects thequality of life of the patients (dyspnoea, low exercise tolerance) andsignificantly reduces their life expectancy. The main risk factorbesides environmental factors is smoking (Kummer F.: Asthma und COPD.Atemw.-Lungenkrkh. 20 (5), 299-302, 1994; Rennard S. I.: COPD: Overviewof definitions, Epidemiology, and factors influencing its development.Chest, 113 (4) Suppl., 235S-241S, 1998) and therefore men are clearlymore often affected than women. As a result of the change in livinghabits and the increase in the number of smokers, this picture, however,will change in future.

The current therapy aims only at the alleviation of the symptoms,without causally intervening in the progression of the disease. The useof long-acting Beta2 agonists (e.g. salmeterol) possibly in combinationwith muscarinergic antagonists (e.g. ipratropium) improves the lungfunction by bronchodilatation and is employed routinely (Norman P.:COPD: New developments and therapeutic opportunities, Drugs NewsPerspect. 11 (7), 431-437, 1998). A large part in the COPD episodes isplayed by bacterial infections, which have to be treated withantibiotics (Wilson R.: The role of infection in COPD, Chest, 113 (4)Suppl., 242S-248S, 1998; Grossman R. F.: The value of antibiotics andthe outcomes of antibiotic therapy in exacerbations of COPD. Chest, 113(4) Suppl., 249S-255S, 1998). The therapy of this disease isunsatisfactory as yet, particularly with respect to the continuousdecrease in lung function. New therapeutic approaches which affectinflammatory mediators, proteases or adhesion molecules could be verypromising (Barnes P. J.: Chronic obstructive disease: new opportunitiesfor drug development, TiPS 10 (19), 415-423, 1998).

Independently of the bacterial infections complicating the disease, achronic inflammation which is dominated by neutrophilic granulocytes isfound in the bronchi. The mediators and enzymes released by neutrophilicgranulocytes, inter alia, have been held responsible for the structuralchanges observed in the airways (emphysema). The inhibition of theactivity of the neutrophilic granulocytes is thus a rational approach toprevent or to slow down progression of COPD (impairment of lung functionparameters). An important stimulus for the activation of thegranulocytes is the pro-inflammatory cytokine TNFα (tumour necrosisfactor). Thus it is known that TNFα stimulates the formation of oxygenradicals by neutrophilic granulocytes (Jersmann, H. P. A.; Rathjen, D.A. and Ferrante A.: Enhancement of LPS-induced neutrophil oxygen radicalproduction by TNFα, Infection and Immunity, 4, 1744-1747, 1998). PDE4inhibitors can very effectively inhibit the release of TNFα from a largenumber of cells and thus suppress the activity of the neutrophilicgranulocytes. The non-specific PDE inhibitor pentoxifylline is able toinhibit both the formation of oxygen radicals and the phagocytosabilityof neutrophilic granulocytes (Wenisch, C.; Zedtwitz-Liebenstein, K.;Parschalk, B. and Graninger W.: Effect of pentoxifylline in vitro onneutrophil reactive oxygen production and phagocytic ability assessed byflow cytometry, Clin. Drug. Invest., 13(2):99-104, 1997).

Various PDE 4 inhibitors are already known. As a matter of priority,these are xanthine derivatives, rolipram analogues or nitraquazonederivatives (general survey in: Karlsson J-A, Aldos D, Phosphodiesterase4 inhibitors for the treatment of asthma, Exp. Opin. Ther. Patents 1997,7:989-1003). Until now, it was not possible to use any of thesecompounds clinically. It had to be established that the known PDE 4inhibitors also have various side-effects such as nausea and emesis,which it was not possible to suppress adequately until now. Thediscovery of new PDE 4 inhibitors with better therapeutic breadth istherefore necessary.

Although indoles have been playing an important part for many years inthe development of new active compounds for various indications, untilnow hydroxyindoles were completely unknown as inhibitors of PDE 4.

DESCRIPTION OF THE INVENTION

The invention relates to substituted hydroxyindoles of the Formula

and their pharmaceutically acceptable salts, wherein

R¹, R⁵ are independently of each other

(i) a C₁₋₁₂ alkyl, straight chain or branched-chain, optionally mono- orpolysubstituted by —OH, —SH, —NH₂, —NHC₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂,—NHC₆₋₁₄ aryl, —N(C₆₋₁₄ aryl)₂, —N(C₁₋₆ alkyl) (C₆₋₁₄ aryl), —NHCOR⁶,—NO₂, —CN, —F, —Cl, Br, —I, —O—C₁₋₆ alkyl, —O—C₆₋₁₄ aryl, —O(CO)R⁶,—S—C₁₋₆ alkyl, —S—C₆₋₁₄ aryl, —SOR⁶, —SO₃H, —SO₂R⁶, —OSO₂C₁₋₆ alkyl,—OSO₂C₆₋₁₄ aryl, —(CS)R⁶, —COOH, —(CO)R⁶, mono-, bi- or tricyclicsaturated or mono- or polyunsaturated carbocycles having from 3 to 14ring members, mono-, bi- or tricyclic saturated or mono- orpolyunsaturated heterocycles having from 5 to 15 ring members and from 1to 6 hetero atoms, which are suitable N, O and S, where the C₆₋₄ arylgroups and the included carbocyclic and heterocyclic substituents canoptionally be mono- or polysubstituted by R⁴.

(ii) —C₂₋₁₂ alkenyl, mono- or polyunsaturated, straight-chain orbranched-chain, optionally mono- or polysubstituted by —OH, —SH, —NH₂,—NHC₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂, —NHC₆₋₁₄ aryl, —N(C₆₋₁₄ aryl)₂, —N(C₁₋₆alkyl)(C₆₋₁₄ aryl), —NHCOR⁶, —NO₂, —CN, —F, —Cl, —Br, —I, —O—C₁₋₆ alkyl,—O—C₆₋₁₄ aryl, —O(CO)R⁶, —S—C₁₋₆ alkyl, —S—C₆₋₁₄ aryl, —SOR⁶, —SO3H,—SO₂R⁶, —OSO₂C₁₋₆ alkyl, —OSO₂C₆₋₁₄ aryl, —(CS)R⁶, —COOH, —(CO)R⁶,mono-, bi- or tricyclic saturated or mono- or polyunsaturatedcarbocycles having from 3 to 14 ring members, mono-, bi- or tricyclicsaturated or mono- or polyunsaturated heterocycles having from 5 to 15ring members and from 1 to 6 heteroatoms, which are suitably N, O and S,where the C₆₋₁₄ aryl groups and the included carbocyclic andheterocyclic substituents for their part can optionally be mono- orpolysubstituted by R⁴,

(iii) mono-, bi- or tricyclic saturated or mono- or polyunsaturatedcarbocycles having from 3 to 14 ring members, optionally mono- orpolysubstituted by —OH, —SH, —NH₂, —NHC₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂,—NHC₆₋₁₄ aryl, —N(C₆₋₁₄ aryl)₂, —N(C₁₋₆ alkyl)(C₆₋₁₄ aryl), —NHCOR⁶,—NO₂, —CN, —F, —Cl, —Br, —I, —O—C₁₋₆ alkyl, —O—C₆₋₁₄ aryl, —O(CO)R⁶,—S—C₁₋₆ alkyl, —S—C₆₋₁₄ aryl, —SOR⁶, —SO₃H, —SO₂R⁶, —OSO₂C₁₋₆ alkyl,—OSO₂C₆₋₁₄ aryl, —(CS)R⁶, —COOH, —(CO)R⁶, mono-, bi- or tricyclicsaturated or mono- or polyunsaturated carbocycles having from 3 to 14ring members, mono-, bi- or tricyclic saturated or mono- orpolyunsaturated heterocycles having from 5 to 15 ring members and from 1to 6 heteroatoms, which are suitably N, O and S, where the C₆₋₁₄ arylgroups and the included carbocyclic and heterocyclic substituents canoptionally be mono- or polysubstituted by R⁴,

(iv) mono-, bi- or tricyclic saturated or mono- or polyunsaturatedheterocycles having from 5 to 15 ring members and from 1 to 6heteroatoms, which are suitably N, O and S, optionally mono- orpolysubstituted by —OH, —SH, —NH₂, —NHC₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂,—NHC₆₋₁₄ aryl, —N(C₆₋₁₄ aryl)₂, —N(C₁₋₆ alkyl)(C₆₋₁₄ aryl), —NHCOR⁶,—NO₂, —CN, —F, —Cl, —Br, —I, —O—C₁₋₆ alkyl, —O—C₆₋₁₄ aryl, —O(CO)R⁶,—S—C₁₋₆ alkyl, —S—C₆₋₁₄ aryl, —SOR⁶, —SO₃H, —SO₂R⁶, —OSO₂C₁₋₆ alkyl,—OSO₂C₆₋₁₄ aryl, —(CS)R⁶, —COOH, —(CO)R⁶, mono-, bi- or tricyclicsaturated or mono- or polyunsaturated carbocycles having from 3 to 14ring members, mono-, bi- or tricyclic saturated or mono- orpolyunsaturated heterocycles having from 5 to 15 ring members and from 1to 6 heteroatoms, which are suitably N, O and S, where the C₆₋₁₄ arylgroups and the included carbocyclic and heterocyclic substituents fortheir part can be optionally mono- or polysubstituted by R⁴, -carbo- orheterocyclic saturated or mono- or polyunsaturated spirocycles havingfrom 3 to 10 ring members, where heterocyclic systems contains from 1 to6 heteroatoms, which are suitably N, O and S, optionally mono- orpolysubstituted by —OH, —SH, —NH₂, —NHC₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂,—NHC₆₋₁₄ aryl, —N(C₆₋₁₄ aryl)₂, —N(C₁₋₆ alkyl)(C₆₋₁₄ aryl), —NHCOR⁶,—NO₂, —CN, —F, —Cl, —Br, —I, —O—C₁₋₆ alkyl, —O—C₆₋₁₄ aryl, —O(CO)R⁶,—S—C₁₋₆ alkyl, —S—C₆₋₁₄ aryl, —SOR⁶, —SO3H, —SO₂R⁶, —OSO₂C₁₋₆ alkyl,—OSO₂C₆₋₁₄ aryl, —(CS)R⁶, —COOH, —(CO)R⁶, mono-, bi- or tricyclicsaturated or mono- or polyunsaturated carbocycles having from 3 to 14ring members, mono-, bi- or tricyclic saturated or mono- orpolyunsaturated heterocycles having from 5 to 15 ring members and from 1to 6 heteroatoms, which are suitably N, O and S, where the C₆₋₁₄ arylgroups and the included carbocyclic and heterocyclic substituents canoptionally be mono- or polysubstituted by R⁴,

R², R³ are hydrogen or —OH, where at least one of the two substituentsmust be —OH;

R⁴ is —H, —OH, —SH, —NH₂, —NHC₁₋₆ alkyl, —N(C,₆ alkyl)₂, —NHC₆₋₁₄ aryl,—N(C₆₋₁₄ aryl)₂, —N(C₁₋₆ alkyl)(C₆₋₁₄ aryl), —NHCOR⁶, —NO₂, —CN, —COOH,—(CO)R⁶, —(CS)R⁶, —F, —Cl, —Br, —I, —O—C₁₋₆ alkyl, —O—C₆₋₁₄ aryl,—O(CO)R⁶, —S—C₁₋₆ alkyl, —S—C₆₋₁₄ aryl, —SOR⁶, —SO₂R⁶.

R⁶ is —H, —NH₂, —NHC₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂, —NHC₆₋₁₄ aryl, —N(C₆₋₁₄aryl)₂, —N(C₁₋₆ alkyl)(C₆₋₁₄ aryl), —O—C₁₋₆ alkyl, —O—C₆₋₁₄ aryl,—S—C₁₋₆ alkyl, —S—C₆₋₁₄ aryl, —C₁₋₁₂ alkyl, straight-chain orbranched-chain, —C₂₋₁₂ alkenyl, mono- or polyunsaturated, straight-chainor branched-chain, -mono-, bi- or tricyclic saturated or mono- orpolyunsaturated carbocycles having from 3 to 14 ring members, -mono-,bi- or tricyclic saturated or mono- or polyunsaturated heterocycleshaving from 5 to 15 ring members and from 1 to 6 heteroatoms, which aresuitably N, O and S;

A is either a bond, or —CH2)_(m)—, —(CH2)_(m)—(CH═CH)_(n)—(CH₂)_(p)—,—(CHOZ)_(m)—, —(C═O)—, —(C═S)—, —(C═N—Z)—, —O—, —S—, —NZ—, where m and pare cardinal numbers from 0 to 3 and n is a cardinal number from 0 to 2,

Z is H, or a C₁₋₁₂ alkyl, straight-chain or branched-chain, C₂₋₁₂alkenyl, mono- or polyunsaturated, straight-chain or branched-chain,mono-, bi- or tricyclic saturated or mono- or polyunsaturatedcarbocycles having from 3 to 14 ring members, mono-, bi- or tricyclicsaturated or mono- or polyunsaturated heterocycles having from 5 to 15ring members and from 1 to 6 heteroatoms, which are suitably N, O and S;

B is either carbon or sulfur, or —(S═O)—;

D is oxygen, sulfur, CH₂ or N—Z, where D can only be S or CH₂ if B iscarbon;

E is a bond, or (CH2)_(m)—, —O—, —S—, —(N—Z)—, where m and Z have thesame meanings as above.

The most suitable compounds of Formula (1) include

N-(3,5-dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl]-2-oxoacetamide;

N-(3,5-dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl]-2-oxoacetamideNa salt;

N-(3,5-dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl]-2-hydroxyacetamide;

N-(pyridin-4-yl)-2-[1-2,6-difluorobenzyl)-5-hydroxyindol-3-yl]-2-oxoacetamide;

N-(3,5-dichloropyridin-4-yl)-2-[1-(2,6-difluorobenzyl)-5-hydroxyindol-3-yl]-2-oxoacetamide;

N-(3,5-dichloropyridin-4-yl)-2-[1-(3-nitrobenzyl)-5-hydroxyindol-3-yl]-2-oxoacetamideNa salt;

N-(3,5-dichloropyridin-4-yl)-2-(1-propyl-5-hydroxyindol-3-yl)-2-oxoacetamide;

N-(3,5-dichloropyridin-4-yl)-2-(1-isopropyl-5-hydroxyindol-3-yl)-2-oxoacetamide;

N-(3,5-dichloropyridin-4-yl)-2-(1-cyclopentylmethyl-5-hydroxyindol-3-yl)-2-oxoacetamide;

N-(2,6-dichlorophenyl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl]-2-oxoacetamide;

N-(2,6-dichloro-4-trifluoromethylphenyl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl)-2-oxoacetamide;

N-(2,6-dichloro-4-trifluoromethoxylphenyl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl)-2-oxoacetamide;

N-(3,5-dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-6-hydroxyindol-3-yl]-2-oxoacetamide;

N-(3,5-dichloropyridin-4-yl)-5-hydroxy-1-(4-methoxybenzyl)indole-3-carboxamide.

The pharmaceutically acceptable salts are obtained in a customary mannerby neutralization of the bases with inorganic or organic acids or byneutralization of the acids with inorganic or organic bases. Possibleinorganic acids are, for example, hydrochloric acid, sulfuric acid,phosphoric acid or hydrobromic acid, organic acids are, for example,carboxylic, sulfo or sulfonic acids such as acetic acid, tartaric acid,lactic acid, propionic acid, glycolic acid, malonic acid, maleic acid,fumaric acid, tannic acid, succinic acid, alginic acid, benzoic acid,2-phenoxybenzoic acid, 2-acetoxybenzoic acid, cinnamic acid, mandelicacid, citric acid, malic acid, salicylic acid, 3-aminosalicylic acid,ascorbic acid, embonic acid, nicotinic acid, isonicotinic acid, oxalicacid, amino acids, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid ornaphthalene-2-sulfonic acid. Possible inorganic bases are, for example,sodium hydroxide solution, potassium hydroxide solution, ammonia, andpossible organic bases are amines, suitably tertiary amines, such astrimethylamine, triethylamine, pyridine, N,N-dimethylaniline, quinoline,isoquinoline, (-picoline, (-picoline, (-picoline, quinaldine orpyrimidine.

In addition, pharmaceutically acceptable salts of the compound ofFormula (1) can be obtained by converting derivatives which havetertiary amino groups into the corresponding quaternary ammonium saltsin a manner known per se by using quaternizing agents. Possiblequaternizing agents are, for example, alkyl halides such as methyliodide, ethyl bromide and n-propyl chloride, but also arylalkyl halidessuch as benzyl chloride or 2-phenylethyl bromide.

Furthermore, the invention of the compounds of Formula (1) which containan asymmetric carbon atom relates to the D form, the L form and D,Lmixtures and, in the case of a number of asymmetric carbon atoms, thediastereomeric forms. Those compounds of Formula (1) which containasymmetric carbon atoms and as a rule are obtained as racemates can beseparated into the optically active isomers in a manner known per se,for example using an optically active acid. However, it is also possibleto employ an optically active starting substance from the start, acorresponding optically active or diastereomeric compound then beingobtained as the final product.

The compounds of the present invention have therapeutically usefulpharmacological properties as inhibitors of the release of TNFα. Thesedisorders include, for example, arthritides including arthritis andrheumatoid arthritis and other arthritic disorders such as rheumatoidspondylitis and osteoarthritis. Further possibilities of theirapplication include the treatment of patients suffering from sepsis,septic shock, gram-negative sepsis, toxic shock syndrome, respiratorydistress syndrome, asthma and other chronic pulmonary disorders, boneresorption diseases or transplant rejection reactions or otherautoimmune disorders, such as lupus erythematosus, multiple sclerosis,glomerulonephritis and uveitis, insulin-dependent diabetes mellitus andchronic demyelinization.

Moreover, the compounds of the present invention can also be employedfor the therapy of infections such as virus and parasite infections, forexample, for the therapy of malaria, infection-related fever,infection-related myalgia, AIDS and cachexia.

The compounds according to the invention are inhibitors ofphosphodiesterase 4 (PDE 4). Therefore, the compounds of Formula (1) andtheir salts, and pharmaceutical preparations which contain thesecompounds or their salts, can be used for the treatment of disorders inwhich inhibition of phosphodiesterase 4 is beneficial.

Thus the compounds according to the invention can be employed asbronchodilators and for asthma prophylaxis. Compounds of Formula (1)also inhibit of the accumulation and activity of eosinophils.Accordingly, the compounds according to the invention can also beemployed in disorders in which eosinophils play a part. These disordersinclude, for example, inflammatory airway disorders such as bronchialasthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis,eczema, allergic anguitis, inflammations mediated by eosinophils such aseosinophilic fasciitis, eosinophilic pneumonia and PIE syndrome(pulmonary infiltration with eosinophilia), urticaria, ulcerativecolitis, Crohn s disease and proliferative skin disorders such aspsoriasis or keratosis.

According to the present invention the compounds of Formula (1) andtheir salts can inhibit both the lipopolysaccharide (LPS)-inducedrelease of TNFα in human blood in vitro, and the LPS-induced pulmonaryneutrophilic infiltration in ferrets and domestic pigs in vivo. All thepharmacologically important properties that were found confirm that thecompounds of Formula (1) and their salts as well as pharmaceuticalpreparations which contain these compounds or their salts can be usedtherapeutically for the treatment of chronic obstructive pulmonarydiseases.

The compounds of the invention also have neuroprotective properties andcan be used for the therapy of diseases in which neuroprotection isbeneficial. Such disorders are, for example, senile dementia(Alzheimer's disease), loss of memory, Parkinson's disease, depression,stroke and intermittent claudication.

Further applications of the compounds of the invention include theprophylaxis and therapy of prostate diseases, such as, for example,benign prostate hyperplasia, pollakiuria, nycturia and for the treatmentof atony of the bladder and of colics caused by kidney stones.

Finally, the compounds according to the invention can also be used forthe inhibition of the development of drug dependence on repeated use ofanalgesics, such as, for example, morphine, and for the reduction of thedevelopment of tolerance on repeated use of these analgesics.

An efficective amount of the compounds according to the invention ortheir salts is used for producing medicaments of the present invention,along with conventional pharmaceutical auxiliaries, carriers andadditives.

The dose of the active compounds can vary depending on factors such asthe route of administration, age and weight of the patient, nature andseverity of the disorders to be treated and similar factors. Therefore,any reference herein to a pharmacologically effective amount of thecompounds of the present invention refers to the aforementioned factors.

The daily dose can be given as an individual dose to be administeredonce or subdivided into two or more daily doses suitably from about0.001 mg to about 100 mg each.

Possible forms of administration include oral, parenteral, intravenous,transdermal, topical, inhalational and intranasal preparations. Foradministration, possible customary pharmaceutical dosage forms includetablets, coated tablets, capsules, dispersible powders, granules,aqueous solutions, aqueous or oily suspensions, syrup, juices and drops.

Solid pharmaceutical forms can contain inert ingredients and carriers,such as, for example, calcium carbonate, calcium phosphate, sodiumphosphate, lactose, starch, mannitol, alginates, gelatin, guar gum,magnesium or aluminium stearates, methylcellulose, talc, highly dispersesalicylic acids, silicone oil, high molecular weight fatty acids (suchas stearic acid), gelatin, agarcagar or vegetable or animal fats andoils, solid high molecular weight polymers (such as polyethyleneglycol); preparations suitable for oral administration can, if desired,contain additional flavorings and/or sweeteners.

Liquid pharmaceutical forms can be sterilized and/or optionally containauxiliaries such as preservatives, stabilizers, wetting agents,penetrating agents, emulsifiers, spreading agents, solubilizers, salts,sugars or sugar alcohols for regulation of the osmotic pressure or forbuffering, and/or viscosity regulators.

Additives of this type include, for example, tartrate and citratebuffers, ethanol, complexing agents (such as ethylenediaminetetraaceticacid and its nontoxic salts). For regulation of the viscosity, possiblehigh molecular weight polymers are those such as, for example, liquidpolyethylene oxide, microcrystalline celluloses,carboxymethylcelluloses, polyvinylpyrrolidones, dextrans or gelatin.Solid carriers include, for example, starch, lactose, mannitol,methylcellulose, talc, highly disperse salicylic acids, high molecularweight fatty acids (such as stearic acid), gelatin, agar-agar, calciumphosphate, magnesium stearate, animal and vegetable fats, solid highmolecular weight polymers such as polyethylene glycol.

Oily suspensions for parenteral or topical application can includevegetable synthetic or semi-synthetic oils such as, for example, liquidC₈₋₂₂ fatty acid esters, for example palmitic, lauric, tridecylic,margaric, stearic, arachidic, myristic, behenic, pentadecanoic,linoleic, elaidic, brassidic, erucic or oleic acid, which are esterifiedwith mono- to C₁₋₆ trihydric alcohols, such as, for example, methanol,ethanol, propanol, butanol, pentanol or their isomers, glycol orglycerol. Fatty acid esters of this type are, for example, commerciallyavailable Miglyols, isopropyl myristate, isopropyl palmitate, isopropylstearate, PEG 6-capric acid, caprylic/capric acid esters of saturatedfatty alcohols, polyoxyethylene glycerol trioleates, ethyl oleate, waxyfatty acid esters such as artificial duck preen gland fat, isopropylcocoate, oleyl oleate, decyl oleate, ethyl lactate, dibutyl phthalate,diisopropyl adipate, polyol fatty acid esters and others. Also suitableare silicone oils of differing viscosities or fatty alcohols such asisotridecyl alcohol, 2-octyldodecanol, cetylstearyl alcohol or oleylalcohol, fatty acids such as, for example, oleic acid. Furthermore,vegetable oils such as castor oil, almond oil, olive oil, sesame oil,cottonseed oil, groundnut oil or soya bean oil can be used.

Possible solvents, gel-forming agents and solubilizers are water orwater-miscible solvents. Those suitable are, for example, alcohols suchas, for example, ethanol or isopropyl alcohol, benzyl alcohol,2-octyldodecanol, polyethylene glycols, phthalates, adipates, propyleneglycol, glycerol, di- or tripropylene glycol, waxes, methylcellosolve,cellosolve, esters, morpholines, dioxane, dimethyl sulphoxide,dimethylformamide, tetrahydrofuran, cyclohexanone etc.

Film-forming agents which can be used are cellulose ethers which candissolve or swell both in water and in organic solvents, such as, forexample, hydroxypropylmethylcellulose, methylcellulose, ethylcelluloseor soluble starches.

Mixed forms between gel- and film-forming agents are also possible.Those used here are especially ionic macromolecules, such as, forexample, sodium carboxymethylcellulose, polyacrylic acid,polymethacrylic acid and its salts, sodium amylopectin semiglycolate,alginic acid or propylene glycol alginate as the sodium salt, gumarabic, xanthan gum, guar gum or carrageenan.

Further formulation auxiliaries which can be employed include glycerol,paraffin of differing viscosities, triethanolamine, collagen, allantoin,novantisolic acid.

The use of surfactants, emulsifiers or wetting agents can also benecessary for formulation, such as, for example, of Na lauryl sulfate,fatty alcohol ether sulfates, di-Na N-lauryl-(-iminodipropionate,polyethoxylated castor oil or sorbitan monooleate, sorbitanmonostearate, polysorbates (e.g. Tween), cetyl alcohol, lecithin,glycerol monostearate, polyoxyethylene stearate, alkylphenyl polyglycolethers, cetyltrimethylammonium chloride or mono-/dialkyl polyglycolether orthophosphoric acid monoethanolamine salts.

Stabilizers such as montmorillonites or colloidal salicylic acids forthe mstabilization of emulsions or for the prevention of the breakdownof the active substances, such as antioxidants, for example tocopherolsor butylhydroxyanisole, or preservatives, such as p-hydroxybenzoic acidesters, can likewise optionally be required for the preparation of thedesired formulations.

Preparations for parenteral administration can be present in separatedose unit forms such as, for example, ampoules or vials. Suitably,solutions of the active compound are used, most suitably aqueoussolutions and especially isotonic solutions, and also suspensions. Theseinjection forms can be made available as finished preparations orprepared only directly before administration by mixing the activecompound, for example the lyophilizate, if appropriate with furthersolid carriers, with the desired solvent or suspending agent.

Intranasal preparations can be present as aqueous or oily solutions oras aqueous or oily suspensions. They can also be present aslyophilizates, which are prepared before administration using thesuitable solvent or suspending agent.

The production, dispensation and scaling of the preparations is carriedout under the conventional antimicrobial and aseptic conditions.

The invention furthermore relates to processes for the preparation ofthe compounds according to the invention.

According to the invention, the compounds of Formula (1) are prepared byconverting compounds of Formula (1), wherein R² or R³ or R² and R³ are—O—R⁷, into the compounds of the invention by removal of R⁷, wherein R⁷is a substituent suitable as a leaving group, such as, for example,alkyl, cycloalkyl, arylalkyl, aryl, heteroaryl, acyl, alkoxycarbonyl,aryloxycarbonyl, aminocarbonyl, N-substituted aminocarbonyl, silyl orsulfonyl groups, and complexing agents, such as, for example, compoundsof boric acid, phosphoric acid and covalently or coordinatively bondedmetals, such as zinc, aluminium or copper.

Particularly suitable reactions for the removal of R⁷ are hydrolysesusing suitable bases, such as, for example, sodium hydroxide solution,potassium hydroxide solution or sodium carbonate or potassium carbonate.

These hydrolyses are suitably used when R⁷ is an acyl, alkoxycarbonyl,aryloxycarbonyl, aminocarbonyl, N-substituted aminocarbonyl, silyl orsulfonyl residue, and a complexing agent, such as, for example,compounds of boric acid, phosphoric acid and coordinatively bondedmetals, such as zinc, aluminium or copper. Particularly suitablereactions for preparing the compounds of the invention for the removalof R⁷ from the compounds in which R⁷ is an alkyl, cycloalkyl, arylalkyl,aryl or heteroaryl residue, are ether cleavages, for example by means ofhydrobromic acid, hydrochloric acid, hydriodic acid, and usingactivating Lewis acids, such as, for example, AlC13, BF3, BBr₃ or LiCl,in each case optionally in the presence of additional activators, suchas, for example, ethane-1,2-dithiol or benzyl mercaptan, and ethercleavages by means of hydrogen, at elevated pressure or at normalpressure, in the presence of a suitable catalyst, such as, for example,a palladium or iridium catalyst.

According to the invention, the compounds of Formula (1) can also beprepared by converting the substructure:

of compounds of Formula (1) by a reaction known per se into othercompounds of Formula (1). Particularly suitable conversion reactionswith compounds of Formula (1) are, for example, when A is —(C═O),reductions to result in A being —(CH—OH)— or A being —CH₂—, by reducingagents known per se, such as, for example, sodium borohydride, or byhydrogenations, which can optionally also be carried outstereoselectively.

Further suitable conversion reactions are the conversion of compounds inwhich D and E are oxygen into substances in which only D is oxygen, butE is —(N—Z)—, where Z has the definition given above.

Exemplary processes show below the preparation of compounds of Formula(1) according to the invention from starting substances of the typedescribed, in which R⁷ is an alkyl, cycloalkyl, arylalkyl, aryl orheteroaryl residue.

EXAMPLE 1N-(3,5-Dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl]-2-oxoacetamide

1.4 g ofN-(3,5-dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-methoxyindol-3-yl]-2-oxoacetamide(3 mmol) is dissolved in 100 ml of dichloromethane. The solution isheated to reflux and treated with a solution of 14 mmol of BBr₃ in 15 mlof dichloromethane with stirring. The reaction mixture is refluxed for 3hours. After cooling, the solution is intensively stirred for 3 hours at20° C. with 200 ml of an aqueous sodium hydrogencarbonate solution. Theproduct crystallizes out, it is isolated, dried at 60° C. andrecrystallized from 80 ml of ethanol.

Yield: 1.1 g (80% of theory); Melting point: 213-214° C.

EXAMPLE 2N-(3,5-Dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl]-2-oxoacetamide

5 g (38 mmol) anhydrous aluminium chloride is introduced into 50 mlethane-1,2, -dithiol. A solution of 4.7 g ofN-(3,5-dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-methoxyindol-3-yl]-2-oxoacetamide(10 mmol) in 50 ml of dichloromethane is added at 0° C. The mixture isstirred at 0° C. for 4 hours. 50 ml of 10% hydrochloric acid is addeddropwise at from 0 to 10° C. with stirring. The crystallizing product isisolated, washed with water and dried at 20° C. A pure product isobtained by recrystallization from ethanol (180 ml).

Yield: 3.1 g (67% of theory); Melting point: 212-214° C.

Exemplary preparative process as follows for compounds of Formula (1)from starting substances of the type described, in which R⁷ is an acyl,alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, N-substitutedaminocarbonyl, silyl or sulfonyl residue:

EXAMPLE 3N-(3,5-Dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl]-2-oxoacetamideNa salt

5 g ofN-(3,5-dichloropyridin-4-yl)-2-[5-acetoxy-1-(4-fluorobenzyl)-indol-3-yl]-2-oxoacetamide(10 mmol) are stirred at 40° C.-50° C. for 1 hour in 50 ml dilute sodiumhydroxide solution. The solution is neutralized with 10% hydrochloricacid while cooling with ice, and is concentrated to dryness. The residueis dissolved in 80 ml acetone and insoluble constituents are removed.The clear solution is treated with a solution of 0.4 g NaOH in 3 ml ofwater and stirred at 20° C. for 2 hours. The crystallized product isisolated, washed with acetone and dried at 60° C.

Yield: 2.44 g (51% of theory); Melting point: 265° C.

An exemplary preparation process follows for compounds of Formula (1)from other compounds of Formula (1).

EXAMPLE 4N-(3,5-Dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl]-2-hydroxyacetamide

1 g ofN-(3,5-dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl]-2-oxoacetamide(1; 2 mmol) are suspended in 75 ml methanol. After addition of asolution of 0.2 g of sodium borohydride in 3 ml dilute sodium hydroxidesolution, the reaction mixture is stirred at 20° C. for 6 hours. Afterthe solvent has been removed by distillation, the residue isrecrystallized from 40 ml ethanol.

Yield: 0.5 g (50% of theory); Melting point: 205-207° C.

Numerous further compounds of Formula (1) can be prepared, as shown inthe Examples and also in the further examples, all summarized in thenext table.

Melting point Ex. R¹ R² R³ R⁴ R⁵ A B D E [° C.] 1 4-Fluorobenzyl —OH —H—H 3,5-Dichloro-4-pyridyl —(C═O)— C O —(N—H)— 215 2 4-Fluorobenzyl —O⁻—H —H 3,5-Dichloro-4-pyridyl —(C═O)— C O —(N—H)— 265 Na⁺ 34-Fluorobenzyl —OH —H —H 3,5-Dichloro-4-pyridyl —(CHOH)— C O —(N—H)—205-207 4 2,6-Difluorobenzyl —OH —H —H 4-Pyridyl —(C═O)— C O —(N—H)—327-329 5 2,6-Difluorobenzyl —OH —H —H 3,5-Dichloro-4-pyridyl —(C═O)— CO —(N—H)— 266-268 6 3-Nitrobenzyl —O⁻ —H —H 3,5-Dichloro-4-pyridyl—(C═O)— C O —(N—H)— 235-238 Na⁺ dec. 7 n-Propyl —OH —H —H3,5-Dichloro-4-pyridyl —(C═O)— C O —(N—H)— 280-282 8 Isopropyl —OH —H —H3,5-Dichloro-4-pyridyl —(C═O)— C O —(N—H)— 245-247 9 Cyclopentylmethyl—OH —H —H 3,5-Dichloro-4-pyridyl —(C═O)— C O —(N—H)— 246-248 104-Fluorobenzyl —OH —H —H 2,6-Dichlorophenyl —(C═O)— C O —(N—H)— 216-21811 4-Fluorobenzyl —OH —H —H 2,6-Dichloro-4- —(C═O)— C O —(N—H)— 199-201trifluoromethylphenyl 12 4-Fluorobenzyl —OH —H —H 2,6-Dichloro-4-—(C═O)— C O —(N—H)— 176-178 trifluoromethoxyphenyl 13 4-Fluorobenzyl —H—OH —H 3,5-Dichloro-4-pyridyl —(C═O)— C O —(N—H)— 212-213 144-Methoxybenzyl —OH —H —H 3,5-Dichloro-4-pyridyl — C O —(N—H)— 239-241

The compounds according to the invention are strong inhibitors ofphosphodiesterase 4 and TNFα release. Their therapeutic potential isconfirmed in vivo, for example, by the inhibition of the asthmaticlate-phase reaction (eosinophilia) in guinea-pigs and by the influencingof the allergen-induced vascular permeability in actively-sensitizedbrown Norway rats.

The PDE 4 inhibiting activity is determined in enzyme preparations ofhuman polymorphonuclear lymphocytes (PMNLs), the PDE 2, 3 and 5 activitywith PDE from human platelets. Human blood was anticoagulated withcitrate. The thrombocyte-rich plasma in the supernatant is separatedfrom the erythrocytes and leucocytes by centrifugation at 700×g for 20minutes at RT. The platelets are lysed by ultrasound and employed in thePDE 3 and PDE 5 assay. For the determination of the PDE 2 activity, thecytosolic platelet fraction is purified on an anion exchange column bymeans of NaCl gradients and the PDE 2 peak is recovered for the assay.The PMNLs for the PDE 4 determination are isolated by a followingdextran sedimentation and subsequent gradient centrifugation usingFicoll-Paque. After a second washing of the cells, the erythrocytesstill contained are lysed in the course of 6 minutes at 4° C. by theaddition of 10 ml of hypotonic buffer (155 mM NH₄Cl, 10 mM NaHCO₃, 0.1mM EDTA, pH 7.4). The still intact PMNLs are washed with PBS a furthertwo times and lysed by means of ultrasound. The supernatant of aone-hour centrifugation at 4° C. at 48,000×g contains the cytosolicfraction of the PDE 4 and is employed for the PDE 4 measurements.

The phosphodiesterase activity is determined with some modificationsaccording to the method described by Thompson et al. (Thompson, W. J.;Appleman, M. M., Assay of cyclic nucleotide phosphodiesterase andresolution of multiple molecular forms of the enzyme, Adv. Cycl. Nucl.Res. 1979, 10, of multiple molecular forms of the enzyme, Adv. Cycl.Nucl. Res. 1979, 10, 69-92).

The reaction mixtures contain 50 mM tris HCl (pH 7.4), 5 mM MgCl₂, theinhibitors in variable concentrations, the corresponding enzymepreparation and also the further components necessary for the detectionof the individual isoenzymes (see below). The reaction is started by theaddition of the substrate 0.5 μM [³H]-cAMP or [³H]-cGMP (about 6000CPM/test). The final volume is 100 ml. Test substances are prepared asstock solutions in DMSO. The DMSO concentration in the reaction mixtureis 1% v/v. At this DMSO concentration, the PDE activity is not affected.After the start of the reaction by means of substrate addition, thesamples are incubated at 37° C. for 30 minutes. The reaction is stoppedby heating the test tubes for 2 minutes at 110° C. The samples remain inthe ice for a further 10 minutes. After the addition of 30 μl of 5-nucleotidase (1 mg/ml, of a snake venom suspension from Crotalusadamanteus) incubation is carried out for 10 minutes at 37° C. Thesamples are stopped on ice, 400 μl each of a mixture ofDowex-water-ethanol (1+1+1) are added, and the samples are well mixedand again incubated on ice for 15 minutes. The reaction vessels arecentrifuged at 3000×g for 20 minutes. 200 μl aliquots of the supernatantare transferred directly to scintillation vessels. After the addition of3 ml of scintillator, the samples are measured in a beta counter.

[³H]-cAMP is used as a substrate for the determination of the PDE 4, 3and 2 activity, [³H]-cGMP for the determination of the PDE 5 activity.The non-specific enzyme activities in each case are determined in thepresence of 100 μM rolipram in the case of PDE 4 and -in the presence of100 μM IBMX in the determination of PDE 3 and 5 and subtracted from thetest values. The incubation batches of the PDE 3 assay contain 10 μMrolipram in order to inhibit possible contamination by the PDE 4. ThePDE 2 is tested using an SPA assay from Amersham. The assay is carriedout in the presence of the activator of PDE 2 (5 μM cGMP).

IC₅₀ values in the range from 10⁻⁹ to 10⁻⁵ M were calculated for thecompounds according to the invention in relation to the inhibition ofphosphodiesterase 4. The selectivity to the PDE types 2, 3 and 5 isfactor 100 to 10,000.

For the determination of the inhibition of TNFα release from cells ofnasal polyps, the experimental arrangement essentially corresponds tothe method described by Campbell, A. M. and Bousquet J (Anti-allergicactivity of H₁-blockers, Int. Arch. Allergy Immunol., 1993, 101,308-310). The starting material is nasal polyps (obtained fromoperation) of patients who have been subjected to surgical treatment.

The tissue is washed with RPMI 1640 and then broken down at 37° C. for 2hours using protease (2.0 mg/ml), collagenase (1.5 mg/ml), hyaluronidase(0.75 mg/ml) and DNAse (0.05 mg/ml) (1 g of tissue to 4 ml of RPMI 1640with enzymes). The cells obtained, a mixture of epithelial cells,monocytes, macrophages, lymphocytes, fibroblasts and granulocytes, arefiltered and washed by repeated centrifugation in nutrient solution,passively sensitized by addition of human IgE and the cell suspension isadjusted to a concentration of 2 million cells/ml in RPMI 1640(supplemented with antibiotics, 10% foetal calf serum, 2 mM glutamineand 25 mM Hepes). This suspension is distributed in 6-well cell cultureplates (1 ml/well). The cells are preincubated for 30 min with the testsubstances in various final concentrations and then stimulated to TNFαrelease by addition of anti-IgE (7.2 μg/ml). The maximum release intothe nutrient medium takes place after about 18 hours. In this period,the cells are incubated at 37° C. and 5% CO₂. The supernatant nutrientmedium is recovered by centrifugation (5 min, 4000 rpm) and stored at−70° C. until cytokine determination. The determination of TNFα in thesupernatant is carried out using so-called sandwich ELISAs (basicmaterial Pharmingen), in which concentrations of the cytokine in therange from 30-1000 pg/ml can be detected.

Cells not stimulated with anti-IgE barely produce TNFα, stimulatedcells, however, secrete large amounts of TNFα, which can be decreased ina dose-dependant manner, for example, by PDE 4 inhibitors. The IC₅₀(concentration at 50% inhibition) is calculated from the percentageinhibition (TNFα release of the cells stimulated with anti-IgE —100%) ofthe tested substances at various concentrations.

For the compounds according to the present invention, IC₅₀ values in therange of 10⁻⁷ to 10⁻⁵ M were determined.

The inhibition of the pulmonary eosinophil infiltration by thesubstances is investigated in an in vivo test of the inhibition of thelate-phase eosinophilia 24 hours after inhalational ovalbumin challengeof actively sensitized guinea-pigs on male Dunkin-Hartley guinea-pigs(200-250 g) actively sensitized against ovalbumin (OVA). Thesensitization is carried out by means of two intraperitoneal injectionsof a suspension of 20 μg of OVA together with 20 mg of aluminiumhydroxide as an adjuvant in 0.5 ml of physiological saline solution peranimal on two successive days. 14 days after the second injection, theanimals are pretreated with mepyramine maleate (10 mg/kg i.p.) in orderto protect them from anaphylactic death. 30 minutes later, the animalsare exposed for 30 sec in a plastic box to an OVA aerosol (0.5 mg/ml)which is generated by a nebulizer driven with compressed air (19.6 kPa)(allergen challenge). Control animals are nebulized with physiologicalsaline solution. 24 hours after the challenge, the animals areanaesthetized with an overdose of ethylurethane (1.5 g/kg of body weighti.p.) and a bronchoalveolar lavage (BAL) is carried out using 2×5 ml ofphysiological saline solution. The BAL fluid is collected, centrifugedat 300 rpm for 10 min and the cell pellet is then resuspended in 1 ml ofphysiological saline solution. The eosinophils in the BAL are countedusing an automatic cell differentiation apparatus (Bayer DiagnosticsTechnicon H1). 2 control groups (nebulization with physiological salinesolution and nebulization with OVA solution) are included in each test.

The percentage inhibition of eosinophilia of the test group treated withsubstance is calculated according to the formula:${\% \quad {inhibition}} = {100 - \frac{100 \times \left( {B - C} \right)}{\left( {A - C} \right)}}$

wherein

A is eosinophils in the control group with OVA challenge and vehicle

B is eosinophils in the group with OVA challenge treated with substance

C is eosinophils in the control group with 0.9% strength NaCl challengeand vehicle

The test substances are administered intraperitoneally or orally as asuspension in 10% polyethylene glycol 300 and 0.5% strength5-hydroxy-ethylcellulose 2 hours before the allergen challenge. Thecontrol groups are treated with the vehicle according to the form ofadministration of the test substance.

The compounds according to the invention were found to inhibitlate-phase eosinophilia by 30% to 80% after intraperitonealadministration of 10 mg/kg and by 40% to 70% after oral administrationof 30 mg/kg. The compounds according to the invention are thusparticularly suitable for the production of drugs for the treatment ofdisorders which are connected with the action of eosinophils.

The effect of allergen-induced vascular permeability was determined onactively sensitized male brown Norway rats. Male brown Norway ratsweighing 280-300 g are actively sensitized on 2 successive days byintraperitoneal injection of a suspension of 1 mg of ovalbumin togetherwith 100 mg of aluminium hydroxide in 1 ml/animal. Three weeks aftersensitization, the rats are anaesthetized with sodium thiopental andfixed in the supine position. A polyethylene catheter was advanced intothe trachea in a backward direction as far as the internal opening ofthe choanas for perfusion of the nasal cavity, so that it was possiblefor the solution to trickle out through the nasal cavities. A shorttracheal catheter was tied into the trachea in an orthograde manner tomake respiration possible. Phosphate-buffered saline solution (PBS) wascontinuously pumped for perfusion through the nasal cavity (0.5 ml/min)using a roller pump and collected by means of a fraction collector.Evans Blue was used as a plasma marker and injected intravenously (1ml/animal each of a 1% strength solution in PBS) through a catheter inthe jugular vein.

Substance administration was carried out topically. Duringadministration, the test substance was added to the perfusion medium(PBS). The nasal mucous membrane was perfused for 30 rain with PDE 4inhibitor-containing solution. Evans Blue was then injected immediatelybefore the start of the perfusion with ovalbumin-containing solution(challenge). After the start of the ovalbumin challenge (10 mg/ml ofovalbumin dissolved in PBS) 15 min fractions were collected every 15 minin the fraction collector over a period of 60 min. The Evans Blueconcentration in the perfusates was measured with a Digiscan photometerat a wavelength of 620 nm. The blank values were automaticallysubtracted in the course of this. The course of action over 60 min wascalculated using an AUC program. The substance action of the preparationgroup was calculated against vehicle controls in %.

IC₅₀ values in the range from 10⁻⁸ to 10⁻⁵ M were determined for thecompounds of the present invention.

The utility of the compounds according to the invention of Formula (I)for the therapy of chronic obstructive pulmonary diseases is confirmedby the inhibition of LPS-induced TNFα release in human blood and by theinhibition of LPS-induced pulmonary neutrophil infiltration in ferretsand domestic pigs, all good animal models.

The stimulation of isolated leucocytes to cytokine release can takeplace in various ways. Lipopolysaccharides (LPSs) are a stimulussuitable for the investigation of TNFα release. LPS is a constituent ofthe bacterial cell walls and is released by killing the bacteria(antibiotics or immune system). LPS particularly stimulates the activityof the phagocytizing leucocytes (tissue macrophages, granulocytes,monocytes) and causes the infiltration of leucocytes from the bloodstream into the affected tissue. A cytokine important for thesemechanisms is TNFα, which is secreted in large amounts by the affectedcells (the monocytes and macrophages are the main source) and initiatesand maintains inflammation alongside other mediators.

For the investigation of the effect on LPS-induced TNFα release, humanblood was obtained from various donors (inhibition of coagulation bymeans of citrate) and diluted 1:5 with RPMI 1640 cell culture medium.The test substances were added to the samples in various concentrationsbefore the LPS challenge. The stimulation of the leucocytes was carriedout 30 min later using lipopolysaccharides (LPS) from Salmonella abortusequi in a final concentration of 10 μg/ml. After incubation of the testbatches for 24 hours at 37° C. and under 5% CO₂ in an incubator, thediluted blood was centrifuged and the TNFα concentration in thecell-free supernatant was measured by means of ELISA.

IC₅₀ values in the range from 10⁻⁷ to 10⁻⁵ M were determined for thecompounds according to the invention. An IC₅₀ value of 0.8 μmol/l, forexample, was determined for the compound as in Example 1. In comparisonwith this, an IC₅₀ value of 7.0 μmol/l was determined with the referencestandard SB 207499.

The inhibition of lipopolysaccharide (LSP)-induced pulmonary neutrophilinfiltration by the substance is investigated in an in vivo test on maleferrets (0.6-2 kg). The experimental animals are anaesthetized withpentobarbital sodium (40 mg/kg of body weight i.p.), placed individuallyinto a closed nebulization box of 5 l capacity and exposed to anultrasonically nebulized aerosol of 0.01% strength LPS(lipopolysaccharide) solution (additionally 0.1% hydroxylamine in PBS)for 10 minutes. The aerosol is generated by a nebulizer driven withcompressed air (0.2 Mpa). Control animals are treated with an aerosol ofphysiological saline solution. The animals are observed during theentire process and removed from the nebulization box after admission offresh air. On inhalation, nebulized LPS immediately induces inflammationof the airways, which is characterized by a massive infiltration ofneutrophilic granulocytes into the lungs of the experimental animals.The neutrophilia achieves its maximum 4 to 6 hours after LPS exposure.In order to be able to measure the number of infiltrated neutrophilicgranulocytes, the animals are anaesthetized with an overdose ofethylurethane (1.5 g/kg of body weight i.p.) 6 hours after LPSprovocation and a bronchoalveolar lavage (BAL) is carried out using 2×10ml of physiological saline solution. The number of cells in the pooledoriginal BAL fluid (100 μl) are determined using the automaticcell-counting apparatus sold by Bayer Diagnostic under the tradedesignation Technicon H1E and the different leucocytes per μl aredifferentiated. In each test, 2 control groups (nebulization withphysiological saline solution or with LPS solution) are included.Substances having anti-inflammatory activity, particularly those whichaffect TNFα release or the function of the neutrophilic granulocytes,inhibit the infiltration of leucocytes. The inhibition of infiltrationis determined by the comparison of the number of infiltrated neutrophilsin untreated experimental animals (with and without LPS provocation).

ID₅₀ values in the range from 1 to 20 mg/kg i.p. were determined for thecompounds according to the invention. The compound of Example 1 wasadministered in doses of 1, 3 and 10 mg/kg i.p. 2 hours before LPSprovocation to up to 3 experimental animals per dose. The neutrophiliain the BAL was inhibited in a dose-dependent manner (18%, 64% and 78%).The ID₅₀ is 2.4 mg/kg i.p. The administration of the selected PDE 4inhibitor RPR-73401 (reference substance) caused an inhibition ofneutrophilia of 49% in the dose 1 mg/kg i.p.

For intrapulmonary administration, the trachea of the animals is openedunder anaesthesia with 40 mg/kg i.p. of pentobarbital sodium, 3%strength, 1.3 ml/kg, a 7 cm-long PVC catheter is tied in and the testsubstances are administered intrapulmonarily in powder form (mixed withlactose to 20 mg/kg) by means of a syringe 2 hours before LPSprovocation. The intrapulmonary administration of Example 1 in doses of1, 3 and 10 mg/kg inhibits LPS-induced neutrophilia in a dose-dependentmanner (43%, 65% and 100%). The ID₅₀ is 1.65 mg/kg i.palm.

Pulmonary neutrophilia can be induced with LPS in domestic pigs in amanner similar to that in the ferret. The animals are anaesthetized withpentobarbital 10 mg/kg i.v., and intubated. Using a bronchoscope, apartial bronchoalveolar lavage is carried out in order to determine theproportion of neutrophilic granulocytes under physiological conditions.The test substance is then administered and the animals inhale anultrasonically nebulized aerosol of 0.03% strength LPS(lipopolysaccharide) solution (additionally 0.1% hydroxylamine in PBS)through the tracheal tube for 20 min. The inhaled LPS induces a reactiveinflammation of the airways and neutrophilic granulocytes infiltrate ona huge scale. The neutrophilia achieves its maximum 4 to 6 hours afterLPS exposure. After 6 hours, the bronchoalveolar lavage is repeated andthe increase in the neutrophil count is determined arithmetically.

Among animal species, the pig is particularly suitable for theseinvestigations, since is has large anatomical and physiologicalsimilarities to man. For the compounds according to the invention,inhibitions of LPS-induced neutrophilia of 20% to 65% were determined onintrapulmonary administration of 10 mg/animal.

The intrapulmonary administration of the compound of Example 1 in thedose 10 mg/animal (about 0.75 mg/kg) inhibited LPS-induced pulmonaryneutrophilia by 51%.

We claim:
 1. The compoundN-(3,5-dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl]-2-oxoacetamide.2. The compoundN-(3,5-dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl]-2-oxoacetamidesodium salt.
 3. A pharmaceutically acceptable salt ofN-(3,5-dichloropyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxyindol-3-yl]-2-oxoacetamide.4. The pharmaceutically acceptable salt of claim 3, wherein the salt isan alkaline earth salt.